Finessing, Extending and Developing an Overview of the Regulation of Ascorbate in plants (FEDORA)
Lead Research Organisation:
UNIVERSITY OF EXETER
Department Name: Biosciences
Abstract
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Technical Summary
FEDORA aims to integrate understanding of the regulation of ascorbate in plants.
1) We will define the molecular mechanism of negative feedback regulation of GGP translation using 5'uORF:luciferase reporters to identify the signal in plant cell and algal cultures.
2) We will use wheat germ in vitro translation to investigate the mechanisms of uORF translation and ribosome stalling that impair translation of GGP using Ribo-seq and modified ChIP to define the association between the uORF peptide and the stalled ribosome(s), leading to structural (low) resolution of the complex.
3) We will investigate how translational control of GGP is set within the boundaries defined by transcriptional control of GGP expression, by comparing proGGP:luciferase reporters with and without mutated uORFs in tomato and Arabidopsis.
4) We will use Arabidopsis lines expressing the proGGP muORF:luciferase reporters to establish a genetic screen for mutations affecting transcriptional regulators of GGP in Arabidopsis.
5) We will test whether transcriptional control by regulators already identified in tomato also operates in Arabidopsis, using T-DNA insertion mutants. We will use genome editing in tomato to develop required mutations in MoneyMaker genetic background.
6) We will investigate how the control of GGP activity impacts flux to ascorbate using targeted MRM analysis by LC-QQQ MS/MS.
7) We will investigate the physiological roles of ascorbate in different plant species by defining the phenotypic consequences of a series of GGP alleles with mis-regulated ascorbate levels, measuring ascorbate and glutathione, leaf number, leaf shape, flowering time, flower number, floral morphology/organ fusion, root biomass, fruit set, fruit weight, shelf life and resistance to necrotrophic pathogens,
8) We will determine whether similar or different phenotypes are observed in Arabidopsis by creating muORFAtGGP1/VTC2 and muORFAtGGP2/VTC5 double mutants by genome editing of Arabidopsis.
1) We will define the molecular mechanism of negative feedback regulation of GGP translation using 5'uORF:luciferase reporters to identify the signal in plant cell and algal cultures.
2) We will use wheat germ in vitro translation to investigate the mechanisms of uORF translation and ribosome stalling that impair translation of GGP using Ribo-seq and modified ChIP to define the association between the uORF peptide and the stalled ribosome(s), leading to structural (low) resolution of the complex.
3) We will investigate how translational control of GGP is set within the boundaries defined by transcriptional control of GGP expression, by comparing proGGP:luciferase reporters with and without mutated uORFs in tomato and Arabidopsis.
4) We will use Arabidopsis lines expressing the proGGP muORF:luciferase reporters to establish a genetic screen for mutations affecting transcriptional regulators of GGP in Arabidopsis.
5) We will test whether transcriptional control by regulators already identified in tomato also operates in Arabidopsis, using T-DNA insertion mutants. We will use genome editing in tomato to develop required mutations in MoneyMaker genetic background.
6) We will investigate how the control of GGP activity impacts flux to ascorbate using targeted MRM analysis by LC-QQQ MS/MS.
7) We will investigate the physiological roles of ascorbate in different plant species by defining the phenotypic consequences of a series of GGP alleles with mis-regulated ascorbate levels, measuring ascorbate and glutathione, leaf number, leaf shape, flowering time, flower number, floral morphology/organ fusion, root biomass, fruit set, fruit weight, shelf life and resistance to necrotrophic pathogens,
8) We will determine whether similar or different phenotypes are observed in Arabidopsis by creating muORFAtGGP1/VTC2 and muORFAtGGP2/VTC5 double mutants by genome editing of Arabidopsis.
Organisations
Publications
Smirnoff N
(2024)
The ascorbate biosynthesis pathway in plants is known, but there is a way to go with understanding control and functions.
in Journal of experimental botany
| Description | We have focussed on the control mechanisms of ascorbate synthesis in plants using tomato and the model plant Arabidopsis. The work focusses on the role of a "key" enzyme in the ascorbate synthesis pathway (GDP-L-galactose phosphorylase, GGP) in controlling ascorbate synthesis and accumulation. Using Arabidopsis plants expressing the luminescent reporter luciferase, we have found that ascorbate and dark supress the expression of the luciferase reporter while light enhances its expression. The repression by ascorbate is dependent on the presence of the 5'-UTR of the GGP mRNA. The 5'-UTR containing a cryptic upstream open reading frame (uORF) which is responsible for ascorbate feedback inhibition of GGP mRNA translation. The characteristics of the uORF are being studied in more detail by our project partner lab (John Innes Centre, Norwich). The uORF repression mechanism avoids over-accumulation of ascorbate in critical plant tissues such as pollen whose development and growth is impaired by elevated ascorbate. High light-induced ascorbate accumulation in Arabidopsis is dependent on increased transcription of the GGP genes in a manner partly dependent on the light signalling transcription factor HY5. The project is revealing that ascorbate content of plant tissues is controlled by an interaction of transcriptional and translational control of the "key" pathway enzyme GGP. |
| Exploitation Route | The project has refined previous observations that a control element (upstream open reading frame, uORF) in the 5'-UTR of the messenger RNA of GDP-L-galactose phosphorylase controls ascorbate (vitamin C) production in plants. We have refined undertanding of this control mechanism opening the way for increasing vitamin C in crop plants by a simple gene editing process. With eventual acceptance of gene editing, we would expect vitamin C enhanced fruit and vegetables to become available. |
| Sectors | Agriculture Food and Drink |